Fluorescent water-soluble probes based on dendritic PEG substituted perylene bisimides: Synthesis, photophysical properties, and live cell images

Article abstract of DOI:10.1039/c2jm30168g

Water-soluble dyes based on dendritic polyethylene glycol (PEG)-substituted perylene bisimides were designed and synthesised. According to the distribution of hydrophilic dendritic PEGs, these dyes can be divided into two classes: (I) head-tail structured

Full text of DOI:10.1039/c2jm30168g

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PAPER
Fluorescent water-soluble probes based on dendritic PEG substituted perylene
bisimides: synthesis, photophysical properties, and live cell images†
a
ac
Hongmei Liu,ac Yongli Wang,b Chenghui Liu,ac Hongxia Li, Baoxiang Gao,
Licui Zhang,a Fuli Bo,a
*
Qianqian Baia and Xinwu Baac
Received 9th January 2012, Accepted 2nd February 2012
DOI: 10.1039/c2jm30168g
Water-soluble dyes based on dendritic polyethylene glycol (PEG)-substituted perylene bisimides were
designed and synthesised. According to the distribution of hydrophilic dendritic PEGs, these dyes can
be divided into two classes: (I) head–tail structured dyes, in which the hydrophobic perylene bisimides
are the heads of the dyes and the hydrophilic dendritic PEG are used as the tails; (II) core–shell
structured dyes, in which the hydrophobic perylene bisimides are encapsulated by the hydrophilic
dendritic PEG. The aggregation behaviour and optical properties of these dyes were investigated. The
two classes of water-soluble perylene bisimides show similar aggregation behaviour, optical and
cytocompatibility properties. With increased dendron PEG generation, the aggregation of the PBI in
an aqueous solution is completely suppressed by the hydrophilic bulky dendritic PEG groups, and the
fluorescence quantum yield increases from 4% to 93%. These dendritic PEG-substituted perylene
bisimides have good cytocompatibility. The head–tail structured dyes show a limited cellular uptake
process. By contrast, the core–shell structured dyes are efficiently internalised by cells and accumulated
in the cytoplasm.
positions.12–14 Although these perylene chromophores showed
Introduction
low to moderate fluorescence quantum yields, their photo-
stability, which is the resistance against photodegradation, is
significantly higher than that of other available fluorophores.
This high photostability is a crucial advantage for long-term
sensitive fluorescence applications.15
Perylene bisimides (PBI) are well-known fluorescent dyes that
display exceptional chemical, thermal, and photochemical
stabilities, as well as high fluorescence quantum yields close to
unity in organic solvents.1,2 Consequently, PBI dyes are widely
applied in various devices, such as organic field-effect transis-
tors,3 organic photovoltaic cells,4 dye lasers5 and optical power
limiters.6 Recently, PBI dyes with photochemical stability have
been established as key chromophores for single molecule spec-
troscopy.7 Single molecule methods also play a growing role in
biophysical studies because they can reveal the dynamics and
mechanistic behaviour that would otherwise be obscured by
ensemble averaging in conventional spectroscopic techniques.8,9
However, PBIs exhibit poor water solubility and very weak
fluorescence in water because of the aggregation of perylene
chromophores.10,11 Therefore, interest in new water-soluble PBI
with high fluorescence quantum yields has continuously
increased over the last few years. Water-solubility has been
achieved by introducing ionic groups on the imide or bay
The largest challenge for PBI chromophores is developing
water-soluble, and non-aggregating analogues that retain their
€
exceptional properties. Recently Wurthner and co-workers have
reported a set of highly water-soluble polyglycerol-dendronised
PBIs with outstanding fluorescence quantum yields in water.16
Furthermore, biocompatible PBIs with high fluorescence in
aqueous media are also highly desired.17 Liu and co-workers
have reported water-soluble probes with good intrinsic cyto-
compatibility, which are intrinsically compatible with living
systems.18
Our group is interested in the development of photochemically
stable and biocompatible dyes with high fluorescence in aqueous
solutions for bioimaging.19 In the current study, the synthesis and
properties of fluorescent water-soluble probes based on dendritic
polyethylene glycol (PEG)-substituted perylene bisimides
(DPPBIs) are presented. Scheme 1 shows the chemical structures
of the DPPBIs. The DPPBIs are prepared with triblock
structures using PBI moieties as fluorescence cores, branched
oligo(glutamic acids) as the scaffold for suppressing the aggre-
gation of the central PBI chromophore, as well as PEG chains as
hydrophilic shells for inducing water solubility and reducing
aCollege of Chemistry and Environmental Science, Hebei University,
Baoding, 071002, P. R. China. E-mail: bxgao@hbu.edu.cn; Fax: +86
3125079317
bSchool of Basic Medicine, Hebei University, Baoding, 071002, P. R. China
cKey Laboratory of Medicinal Chemistry and Molecular Diagnosis,
Ministry of Education, Hebei University, Baoding, 071002, P. R. China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c2jm30168g
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MTT (10 mL, 5 mg mLꢂ1) solution in a culture medium was
added into each well. The MTT medium solution was carefully
removed after 3 h incubation in the incubator. DMSO (100 mL)
was then added into each well and the wells were gently shaken
for 10 min at room temperature to dissolve all the precipitate
formed. The absorbance of MTT at 490 nm was monitored by an
ELX-800 microplate reader (ELISA Reader). Cell viability was
expressed by the ratio of the absorbance of the cells incubated
with PEPBI solution. Each result is an average of data from six
wells; 100% viability was determined using untreated cells.
Live cell imaging. HeLa cells were grown in Minimum Essen-
tial Medium Eagle (MEM medium, Sigma) in a 35 mm glass
bottomed poly-D-lysine coated Petri-dish for at least 24 h to
enable adherence to the bottom. The MEM medium was then
replaced by the G2 solution (1.0 ꢁ 10ꢂ5 mol Lꢂ1) for 1 h, and then
washed three times with PBS buffer. CLSM images were
obtained using confocal laser scanning microscopy (Olympus
Fluoview FV1000).
Scheme 1 Chemical structures of the DPPBIs.
cytotoxicity. Based on the distribution of the hydrophilic
dendritic PEG, these DPPBI compounds can be divided into two
classes. The first comprises the head–tail structured dyes: AG1,
AG2 and AG3, in which the hydrophobic PBIs are the heads of
the dyes and the hydrophilic dendritic PEGs are used as tails. The
second comprises the core–shell structured dyes G1, G2 and G3,
in which the hydrophobic PBIs are encapsulated by the hydro-
philic dendritic PEGs.
Synthesis and characterization of DPPBIs
Compound AG1. The synthesis procedures for all DPPBIs are
outlined in Scheme 2. 2,5,8,12,15,18-Hexaoxa-10-nonadecan-
amine (0.59 g, 2.0 mmol), 3,4,9,10-perylenetetracarboxylic dia-
nhydride (0.83 g, 1.8 mmol), 15 g imidazole and 135 mg
Zn(OAc)2 (0.6 mmol) were heated at 120 ꢀC for 4 h. The reaction
mixture solidified upon cooling and was dissolved in 2 M HCl.
DCM was added and the phases were separated. The water phase
was extracted thoroughly with DCM and then the combined
Experimental
General information
Perylenetetracarboxylic acid dianhydride was purchased from
Liaoning Liangang Pigment and Dyestuff Chemicals Co. Ltd.
L-Aspartic acid and N-carbobenzyloxy-L-aspartic acids were
purchased from Yangzhou Baosheng Bio-chemical Co. Ltd.
N-(3-Dimethylaminopropyl)-N0-ethylcarbodiimide hydrochlo-
ride (EDCI), 4-dimethylaminopyridine and 1-hydroxybenzo-
trizole were purchased from Shanghai Medpep Co. Ltd.
Imidazole and all solvents were purchased from Sinopharm
Chemical Reagent Co. Ltd., and used without any further
purification. Compound 1, 3, 4 and 6 were synthesized according
to the reported methods.20,21 Solvents used for precipitation and
column chromatography were distilled under a normal atmo-
1
ꢀ
sphere. The H-NMR spectra were recorded at 20 C on a 600
MHz NMR spectrometer (Bruker). Chemical shifts are reported
in ppm at room temperature using CDCl3 as solvent and tetra-
methylsilane as internal standard unless indicated otherwise.
Abbreviations used for splitting patterns are s ¼ singlet, d ¼
doublet, t ¼ triplet, qui ¼ quintet, m ¼ multiplet. Mass spectra
were carried out using MALDI-TOF/TOF matrix assisted laser
desorption ionization mass spectrometry with AutoflexIII
Smartbeam (Bruker Daltonics Inc.). UV/Vis spectra were
recorded on a Shimadzu WV-2550 spectrophotometer. Fluores-
cence spectra were recorded on a Shimadzu RF-5301 fluores-
cence spectrophotometer.
Cytotoxicity test. MTT assays were performed to assess the
metabolic activity of HeLa cells. HeLa cells were seeded in 96-
well plates (Costar, IL, USA) at an intensity of 2 ꢁ 104 cells
mLꢂ1. After 36 h incubation, the medium was replaced by PEPBI
solution at the concentration of 1.0 ꢁ 10ꢂ5 mol Lꢂ1, and the cells
were then incubated for 24 h. After the designated time intervals,
the wells were washed twice with PBS buffer and freshly prepared
Scheme 2 Synthesis of DPPBIs.
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organic layers were dried over Na2SO4 and concentrated. Lastly,
imidazole-free material was obtained by column chromatog-
raphy on silica gel to give a deep-red solid (970 mg, 73%).
1H-NMR (600 MHz, CDCl3): d 0.95 (m, 6H), 1.96 (m, 2H),
2.28 (m, 2H),3.28 (s, 6H), 3.42 (m, 4H), 3.56 (m, 4H), 3.62 (m,
4H), 3.66 (m, 2H), 3.74 (m, 2H), 3.98 (m,2H), 4.20 (m, 2H),
5.08 (m, 1H), 5.71 (m, 1H), 8.51 (d, J ¼ 7.8 Hz, 2H), 8.59 (d, J ¼
8.4 Hz,4H), 8.65 (d, J ¼ 7.8 Hz, 2H); MALDI-TOF MS m/z
calcd for C42H46N2O10: 738.82, found: 761.3 [M + Na]+.
in molten imidazole with zinc acetate as a catalyst. The
2,5,8,12,15,18-hexaoxa-10-nonadecanamine 4, a key interme-
diate, was obtained according to literature procedures.21 The
reaction of
4 with Cbz-protected L-aspartic acid yielded
compound 5, and then followed by the removal of the Cbz
group by catalytic hydrogenation using Pd/C. AG2 and AG3
were prepared by DCC-coupling with the unsymmetric PBI 3
and 2,5,8,12,15,18-hexaoxa-10-nonadecanamine, compound 6.
These unsymmetric dendritic PBIs were purified by a combi-
nation of column chromatography on silica gel and size-
exclusion chromatography (Biobeads, SX-3, THF). These
DPPBIs were fully characterized by 1H-NMR spectroscopy,
and matrix assisted laser desorption/ionization mass spec-
trometry. All spectroscopy can be found in the ESI†.
Compound AG2. Compound 3 (115 mg, 0.2 mmol), EDCI
(96 mg, 0.5 mmol), and HOBt (76 mg, 0.5 mmol) were dissolved in
40 mL dry DMF. The mixture was cooled to 0 ꢀC and stirred for
0.5 h before removal of the ice bath. Subsequently, compound 4
(148 mg, 0.5 mmol) was added. The reaction was stirred for 36 h at
room temperature. After that, the solvent DMF was evaporated
under reduced pressure. The solid mixture was dried under
vacuum at 60 ꢀC, and then purified by column chromatography
on silica gel to yield a pure red product (185 mg, 82%).
Aggregation behaviour and optical properties
In the hydrophobic toluene solvent, compounds AG1, AG2,
AG3, and G1 show non-aggregation behaviour. Their absorption
maximum appears at 529 nm, along with two higher transitions
located at 495 and 462 nm, which is a typical for the dissolved
PBI molecule (Fig. 1A). With increased dendron PEG genera-
tion, a blue shift of the absorption maximum of G2 and G3 is
observed (Fig. 1C). The intensity reversal observed for the
0–0 and 0–1 bands indicates that the Franck–Condon factors
now favour the higher (0–1) excited state,22 suggesting the
formation of H-type cofacial p–p stacking in the hydrophobic
solvent. The transformation from hydrophobic to hydrophilic
was further demonstrated by the UV-vis absorption spectra of
DPPBIs in water (Fig. 1B and Fig. 1D). Compound AG1 is not
soluble in water, whereas the others have good water solubility.
In the aqueous solution, the absorption maximum of compounds
AG2 and AG3 appears at 495 nm (Fig. 1B), suggesting strong
aggregation behaviour. However, compound G3 has a non-
aggregation UV-vis absorption with the absorption maximum at
529 nm (Fig. 1D), which is the typical absorption of dissolved
PBI molecules. This result suggests that the PEG chains function
as hydrophilic shells for improving the hydrophilic properties of
the perylene dyes.
1H-NMR (600 MHz, CDCl3): d 0.95 (m, 6H), 1.97 (m, 4H),
2.27 (m, 1H), 2.92 (m, 1H), 3.32 (m, 12H), 3.44–3.62 (m, 40H),
4.16 (m, 1H), 4.29 (m, 1H), 5.06 (m, 1H), 6.11 (m, 1H), 6.76 (d,
J ¼ 8.4 Hz, 1H), 7.39 (d, J ¼ 8.4 Hz, 1H), 8.51 (d, J ¼ 8.4 Hz,
2H), 8.53 (d, J ¼ 7.8 Hz, 2H), 8.59 (d, J ¼ 7.8 Hz, 2H), 8.63 (d,
J ¼ 7.8 Hz, 2H); MALDI-TOF MS m/z calcd for C59H78N4O18:
1131.27, found: 1153.5 [M + Na]+.
Compound AG3. Compound 3 (70 mg, 0.12 mmol), EDCI
(58 mg, 0.3 mmol), and HOBt (46 mg, 0.3 mmol) were dissolved
in 25 mL dry DMF. The mixture was cooled to 0 ꢀC and stirred
for 0.5 h before removal of the ice bath. Subsequently,
compound 6 (412 mg, 0.6 mmol) was added. The reaction was
stirred for 72 h at room temperature. After that, the solvent
DMF was evaporated under reduced pressure. The solid mixture
was dried under vacuum at 60 ꢀC, and then purified by
column chromatography on silica gel to yield a red product
(180 mg, 78%).
1H-NMR (600 MHz, CDCl3): d 0.91 (m, 6H), 1.93 (m, 2H),
2.15 (m, 2H), 2.55 (m, 3H), 2.84 (m, 3H), 3.32 (m, 12H), 3.42–
3.66 (m, 90H), 4.13 (m, 4H), 4.67 (m, 2H), 5.03 (m, 1H), 6.15 (m,
1H), 6.60 (d, J ¼ 8.4 Hz, 2H), 7.13 (d, J ¼ 8.4 Hz, 1H), 7.76 (d,
J ¼ 8.4 Hz, 2H), 7.90 (d, J ¼ 8.4 Hz, 1H), 8.52–8.61 (m, 8H);
MALDI-TOF MS m/z calcd for C93H142N8O34: 1916.16, found:
1937.9 [M + Na]+.
Results and discussion
Synthesis
The symmetric PBIs dendronized with first-, second-, and
third-generation dendrons were synthesized according to the
reported methods.19a The unsymmetric dendritic PBIs were
synthesized similar to the route for symmetric PBIs, where the
swallow-tailed perylene monoimide monoanhydride starting
compound 1 was obtained by the basic saponification of
symmetric the N,N0-bis(ethylpropyl)perylene-3,4:9,10-tetra-
carboxylic diimide. The compound 1 was condensed with
L-aspartic acid or the swallow-tailed 2,5,8,12,15,18-hexaoxa-10-
nonadecanamine, 4, to yield the unsymmetric PBI 3 and AG1
Fig. 1 UV-Vis absorption spectra of DPPBIs in toluene (left) and water
(right) with a concentration of 1.0 ꢁ 10ꢂ4 M.
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Table 1 Fluorescence data for the DPPBIsa
Solvent
Water
AG1
AG2
AG3
G1
G2
G3
b
lmaxpl/nm
Fc (%)
lmaxpl/nm
Fc (%)
—
—
547
5
542
68
549
70
544
73
547
4
542
75
550
65
540
48
552
93
544
26
b
Toluene
541
95
a
b
c
10ꢂ5 mol Lꢂ1, excitation wavelength is 492 nm. Insoluble. N,N0-
Dicyclohexyl-perylene-3,4:9,10-tetracarboxylic
chloroform was used as reference (F ¼ 100%).
acid
bisimide
in
aqueous and toluene solutions show a broad band with lmax at
540 nm to 545 nm. In the hydrophobic toluene solvent, AG1 has
the highest fluorescence quantum yield (95%). After introducing
more hydrophobic PEG chains on the end of the imide, the
fluorescence quantum yield drops from 95% (AG1) to 26% (G3).
The aggregation behaviour of the DPPBIs in the toluene solution
is responsible for the decrease in the fluorescence quantum yield.
In contrast, AG2 and G1 in the aqueous solution show low
fluorescence quantum yields. With more hydrophobic PEG
chains on the end of the imide, the fluorescence quantum yield
increases from 4% (G1) to 93% (G3). It is further demonstrated
that the aggregation of the DPPBIs in the aqueous solution is
completely suppressed by the hydrophilic bulky dendritic PEG
groups.
Fig. 2 Concentration-dependent UV-vis spectra of the DPPBIs.
The aggregation behavior of DPPBIs in solution was investi-
gated via concentration-dependent UV-vis absorption spectros-
copy (Fig. 2). In a low-concentration aqueous solution (1.0 ꢁ
10ꢂ6 mol Lꢂ1), the absorption bands of DPPBIs show
a maximum absorption at 533 nm, which can be attributed to the
p–p transition of the PBI monomer. When the aqueous solution
concentration increases from 1.0 ꢁ 10ꢂ6 to 1.0 ꢁ 10ꢂ4 mol Lꢂ1
,
the absorption coefficients of AG2 and AG3 dramatically
decrease by 62% and 57%, respectively. A blue shift of the
absorption maximum to 491 nm is observed, implying the
formation of H-type cofacial p–p stacking in a high-concentra-
tion aqueous solution. By contrast, in the hydrophobic
toluene solvent with concentrations from 1.0 ꢁ 10ꢂ6 to 1.0 ꢁ
10ꢂ4 mol Lꢂ1, the absorption coefficient of the hydrophilic
compound G3 dramatically decreases by 60%. However, in an
aqueous solution with the same concentration range, the
extinction coefficient (0–0 transition) of G3 does not change. It
suggests that the aggregation of the PBIs is completely sup-
pressed by the hydrophilic bulky dendritic PEG groups.
Cytocompatibility properties and live cell images
To demonstrate the potential utility of the DPPBIs for cellular
imaging, their cytocompatibility or cytotoxicity must be assessed.
The cytocompatibility of the DPPBIs was evaluated for HeLa
cells using an MTT cell-viability assay. Fig. 4 summarizes the
viability of HeLa cells after being cultured with the DPPBI
solutions at a concentration of 5.0 ꢁ 10ꢂ5 mol Lꢂ1 for 24 h. These
compounds showed very low cytotoxicity (over 90% viability)
within 24 h of incubation time. This result indicates that the PEG
encapsulation of perylene bisimides has high cytocompatibility.
The exceptionally low cytotoxicity is ascribed to the PEG chains,
which protect the dyes from nonspecifically interacting with the
extracellular proteins.23
Fig. 3 shows the fluorescence emission spectra of these
DPPBIs in aqueous and toluene solutions. The wavelength of the
emission maxima and fluorescence quantum yields are given in
Table 1. The fluorescence emission spectra of these DPPBIs in
Fig. 3 Fluorescence emission spectra of the DPPBIs in water (top) and
Fig. 4 In vitro viability of HeLa cells treated with the DPPBI solution at
1.0 ꢁ 10ꢂ5 mol Lꢂ1 for 24 h.
in toluene (bottom).
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Fig. 5 Bright-field images of HeLa cells stained by AG2 (A), AG3 (C),
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Conclusions
The synthesis and properties of fluorescent water-soluble probes
based on dendritic PEG-substituted perylene bisimides are
reported. These dyes can be divided into two classes, namely,
head–tail and core–shell structured dyes. The two classes of
water-soluble PBIs show similar aggregation behaviour, optical,
and cytocompatibility properties. With increased dendron PEG
generation, the aggregation of the PBIs in aqueous solution is
completely suppressed by the hydrophilic bulky dendritic PEG
groups, and the fluorescence quantum yield increases from 4% to
93%. High fluorescence quantum yields, good water solubility,
photostability, and low cytotoxicity make these DPPBIs excel-
lent candidates as a new class of probes for live cell imaging. The
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This work was supported by the National Natural Science
Foundation of China (No. 20804012), Hebei University Natural
Science Funds for Distinguished Young Scholar (J2010-01), the
Key Project of Chinese Ministry of Education (No. 20100015),
the Natural Science Foundation of Hebei Province (No.
B2009000169).
€
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